Effect of condensed tannins from tropical legumes on the activity of fibrolytic enzymes from the rumen fungus Neocallimastyx hurleyensis

A study was conducted to assess the effect of condensed tannins on the activity of fibrolytic enzymes from the anaerobic rumen fungus, Neocallimastix hurleyensis and a recombinant ferulic acid esterase (FAE) from the aerobic fungus Aspergillus niger. Condensed tannins were extracted from the tropical legumes Desmodium ovalifolium, Flemingia macrophylla, Leucaena leticocephala, Leucaena pallida, Calliandra calothyrsus and Clitoria fairchildiana and incubated in fungal enzyme mixtures or with the recombinant FAE. In most cases, the greatest reductions in enzyme activities were observed with tannins purified from D. ovalifolium and F macrophylla and the least with tannins from L leucocephala. Thus, whereas 40 mu g ml(-1) of condensed tannins from C. calothyrsus and L. leucocephala were needed to halve the activity of N. hurleyensis carboxymethylcellulase (CMCase), just 5.5 mu g ml(-1) of the same tannins were required to inhibit 50% of xylanase activity. The beta-D-glucosidase and beta-D-Xylosidase enzymes were less sensitive to tannin inhibition and concentrations greater than 100 mu g ml(-1) were required to reduce their activity by 50%. In other assays, the inhibitory effect of condensed tannins when added to incubation mixtures containing particulate substrates (the primary cell walls of E arundinacea) or when bound to these substrate was compared. Substrate-associated tannins were more effective in preventing fibrolytic activities than tannins added directly to incubations solutions. It was concluded that condensed tannins from tropical legumes can inhibit fibrolytic enzyme activities, although the extent of the effect was dependent on the tannin, the nature of its association with the substrate and the enzyme involved. (c) 2005 Elsevier Inc. All rights reserved.

G-protein-coupled-receptor-mediated presynaptic inhibition in the cerebellum

Throughout the central nervous system a dominant form of inhibition of neurotransmitter release from presynaptic terminals is mediated by G-protein-coupled receptors (GPCRs). Neurotransmitter release is typically induced by action potentials (APs), but can also occur spontaneously. Presynaptic inhibition by GPCRs has been associated with modulation of voltage-dependent ion channels. However, electrophysiological recordings of spontaneous, AP-independent (so-called ‘miniature’) postsynaptic events reveal an additional, important form of GPCR-mediated presynaptic inhibition, distinct from effects on ionic conductances and consistent with a direct action on the vesicle release machinery. Recent studies suggest that such miniature events might be of physiological relevance not only in signalling but also in development. In the cerebellum, neurotransmitter release onto Purkinje cells occurs by AP-dependent and AP-independent pathways. Here, I focus on inhibitory synapses between interneurons and Purkinje cells, which are subject to strong, identifiable regulation by endogenous GPCR agonists, to consider mechanisms of GPCR-mediated presynaptic inhibition.

Pea-barley intercropping for efficient symbiotic N-2-fixation, soil N acquisition and use of other nutrients in European organic cropping systems

Complementarity in acquisition of nitrogen (N) from soil and N-2-fixation within pea and barley intercrops was studied in organic field experiments across Western Europe (Denmark, United Kingdom, France, Germany and Italy). Spring pea and barley were sown either as sole crops, at the recommended plant density (P100 and B100, respectively) or in replacement (P50B50) or additive (P100B50) intercropping designs, in each of three cropping seasons (2003-2005). Irrespective of site and intercrop design, Land Equivalent Ratios (LER) between 1.4 at flowering and 1.3 at maturity showed that total N recovery was greater in the pea-barley intercrops than in the sole Crops Suggesting a high degree of complementarity over a wide range of growing conditions. Complementarity was partly attributed to greater soil mineral N acquisition by barley, forcing pea to rely more on N-2-fixation. At all sites the proportion of total aboveground pea N that was derived from N-2-fixation was greater when intercropped with barley than when grown as a sole crop. No consistent differences were found between the two intercropping designs. Simultaneously, the accumulation Of Phosphorous (P), potassium (K) and sulphur (S) in Danish and German experiments was 20% higher in the intercrop (P50B50) than in the respective sole crops, possibly influencing general crop yields and thereby competitive ability for other resources. Comparing all sites and seasons, the benefits of organic pea-barley intercropping for N acquisition were highly resilient. It is concluded that pea-barley intercropping is a relevant cropping strategy to adopt when trying to optimize N-2-fixation inputs to the cropping system. (C) 2009 Elsevier B.V. All rights reserved.

The biocontrol fungus Pochonia chlamydosporia shows nematode host preference at the infraspecific level

A RAPD-PCR assay was developed and used to test For competitive variability in growth of the nematode biological control fungus Pochonia chlamydosporia. Saprophytic competence in soil with or without tomato plants was examined in three isolates of the fungus: RES 280 (J), originally isolated from potato cyst nematode (PCN) cysts; RES 200 (1) and RES 279 (S), both originally isolated from root knot nematode (RKN) eggs. Viable counts taken at 70 d indicated that I was the best saprophyte followed by S, with J the poorest. RAPD-PCR analysis of colonies from mixed treatments revealed that there was a cumulative effect of adding isolates to the system. This Suggested that the isolates did not interact and that they may occupy separate niches in soil and the rhizosphere. To investigate parasitic ability, soils were seeded with two isolates of the fungus: J and S, singly or in combination. Tomato or potato plants were grown in these soils; free of nematodes, or inoculated with PCN or RKN, and incubated for 77 d. The abundance of the PCN isolate J in PCN cysts was significantly greater than that of the RKN isolate S but in RKN egg masses, S was significantly more abundant than J. RAPD-PCR analysis of colonies from mixed treatments confirmed that J was more abundant than S ill PCN cysts whereas the converse was observed on RKN egg masses. This substantiates the phenomenon of nematode host preference at the infraspecific level of P. chlamydosporia and highlights its relevance for biological control of plant parasitic nematodes.

Inhibition of human mesangial cell proliferation by targeting T-Type calcium channels

Background: Aberrant glomerular mesangial cell (MC) proliferation is a common finding in renal diseases. T-type calcium channels (T-CaCN) play an important role in the proliferation of a number of cell types, including vascular smooth muscle cells. The hypothesis that T-CaCN may play a role in the proliferation of human MC was investigated. Methods: The presence of T-CaCN in primary cultures of human MC was examined using voltage clamping and by RT-PCR. The effect of calcium channel inhibitors, and of siRNA directed against the Cav3.2 T-CaCN isoform, on MC proliferation was assessed using the microculture tetrazolium assay and nuclear BrdU incorporation. Results: Human MC express only the Cav3.2 T-CaCN isoform. Co-incubation of MC with a T-CaCN inhibitor (mibefradil, TH1177 or Ni2+) results in a concentration-dependent attenuation of proliferation. This effect cannot be attributed to direct drug-induced cytotoxicity or apoptosis and is not seen with verapamil, an L-type channel blocker. Transfection of MC with siRNA results in knockdown of T-CaCN Cav3.2 mRNA and a clear attenuation of MC proliferation. Conclusions: These results demonstrate for the first time an important role for T-CaCN in human MC proliferation. This could potentially lead to a novel therapy in the treatment of proliferative renal diseases.

Fas ligand-induced apoptosis is regulated by nitric oxide through the inhibition of fas receptor clustering and the nitrosylation of protein kinase Cepsilon

Apoptosis induced by the death-inducing ligand FasL (CD95L) is a major mechanism of cell death. Trophoblast cells express the Fas receptor yet survive in an environment that is rich in the ligand. We report that basal nitric oxide (NO) production is responsible for the resistance of trophoblasts to FasL-induced apoptosis. In this study we demonstrate that basal NO production resulted in the inhibition of receptor clustering following ligand binding. In addition NO also protected cells through the selective nitrosylation, and inhibition, of protein kinase Cepsilon (PKCepsilon) but not PKCalpha. In the absence of NO production PKCepsilon interacted with, and phosphorylated, the anti-apoptotic protein cFLIP. The interaction is predominantly with the short form of cFLIP and its phosphorylation reduces its recruitment to the death-inducing signaling complex (DISC) that is formed following binding of a death-inducing ligand to its receptor. Inhibition of cFLIP recruitment to the DISC leads to increased activation of caspase 8 and subsequently to apoptosis. Inhibition of PKCepsilon using siRNA significantly reversed the sensitivity to apoptosis induced by inhibition of NO synthesis suggesting that NO-mediated inhibition of PKCepsilon plays an important role in the regulation of Fas-induced apoptosis.

Non-genomic signalling of the retinoic X receptor through inhibition of Gq signalling in human platelets

Retinoid X receptors (RXRs) are important transcriptional nuclear hormone receptors, acting as either homodimers or the binding partner for at least one fourth of all the known human nuclear receptors. Functional nongenomic effects of nuclear receptors are poorly understood; however, recently peroxisome proliferator-activated receptor (PPAR) gamma, PPARbeta, and the glucocorticoid receptor have all been found active in human platelets. Human platelets express RXRalpha and RXRbeta. RXR ligands inhibit platelet aggregation and TXA(2) release to ADP and the TXA(2) receptors, but only weakly to collagen. ADP and TXA(2) both signal via the G protein, Gq. RXR rapidly binds Gq but not Gi/z/o/t/gust in a ligand-dependent manner and inhibits Gq-induced Rac activation and intracellular calcium release. We propose that RXR ligands may have beneficial clinical actions through inhibition of platelet activation. Furthermore, our results demonstrate a novel nongenomic mode for nuclear receptor action and a functional cross-talk between G-protein and nuclear receptor signaling families.

Inhibition of coxsackie B virus infection by soluble forms of its receptors: Binding affinities, altered particle formation, and competition with cellular receptors

We previously reported that soluble decay-accelerating factor (DAF) and coxsackievirus-adenovirus receptor (CAR) blocked coxsackievirus 133 (CVB3) myocarditis in mice, but only soluble CAR blocked CVB3-mediated pancreatitis. Here, we report that the in vitro mechanisms of viral inhibition by these soluble receptors also differ. Soluble DAF inhibited virus infection through the formation of reversible complexes with CVB3, while binding of soluble CAR to CVB induced the formation of altered (A) particles with a resultant irreversible loss of infectivity. A-particle formation was characterized by loss of VP4 from the virions and required incubation of CVB3-CAR complexes at 37 degrees C. Dimeric soluble DAF (DAF-Fc) was found to be 125-fold-more effective at inhibiting CVB3 than monomeric DAF, which corresponded to a 100-fold increase in binding affinity as determined by surface plasmon resonance analysis. Soluble CAR and soluble dimeric CAR (CAR-Fc) bound to CVB3 with 5,000- and 10,000-fold-higher affinities than the equivalent forms of DAF. While DAF-Fc was 125-fold-more effective at inhibiting virus than monomeric DAF, complement regulation by DAF-Fc was decreased 4 fold. Therefore, while the virus binding was a cooperative event, complement regulation was hindered by the molecular orientation of DAF-Fc, indicating that the regions responsible for complement regulation and virus binding do not completely overlap. Relative contributions of CVB binding affinity, receptor binding footprint on the virus capsid, and induction of capsid conformation alterations for the ability of cellular DAF and CAR to act as receptors are discussed.

Follistatin complexes Myostatin and antagonises Myostatin-mediated inhibition of myogenesis

Follistatin is known to antagonise the function of several members of the TGF-beta family of secreted signalling factors, including Myostatin, the most powerful inhibitor of muscle growth characterised to date. In this study, we compare the expression of Myostatin and Follistatin during chick development and show that they are expressed in the vicinity or in overlapping domains to suggest possible interaction during muscle development. We performed yeast and mammalian two-hybrid studies and show that Myostatin and Follistatin interact directly. We further show that single modules of the Follistatin protein cannot associate with Myostatin suggesting that the entire protein is required for the interaction. We analysed the interaction kinetics of the two proteins and found that Follistatin binds Myostatin with a high affinity of 5.84 x 10(-10) M. We next tested whether Follistatin suppresses Myostatin activity during muscle development. We confirmed our previous observation that treatment of chick limb buds with Myostatin results in a severe decrease in the expression of two key myogenic regulatory genes Pax-3 and MyoD. However, in the presence of Follistatin, the Myostatin-mediated inhibition of Pax-3 and MyoD expression is blocked. We additionally show that Myostatin inhibits terminal differentiation of muscle cells in high-density cell cultures of limb mesenchyme (micromass) and that Follistatin rescues muscle differentiation in a concentration-dependent manner. In summary, our data suggest that Follistatin antagonises Myostatin by direct protein interaction, which prevents Myostatin from executing its inhibitory effect on muscle development. (C) 2004 Elsevier Inc. All rights reserved.

Combining competition from Lolium perenne and an insect-fungus combination to control Rumex obtusifolius seedlings

Although adult Rumex obtusifolius are problematic weeds, their seedlings are poor competitors against Lolium perenne, particularly in established swards. We investigated the possibility of using this weakness to augment control of R. obtusifolius seedlings with combinations of Gastrophysa viridula (Coleoptera: Chrysomelidae) and the rust fungus Uromyces rumicis. Rumex obtusifolius seedlings were grown in competition with L. perenne sown at different rates and times after R. obtusifolius: they competed successfully with L. perenne when sown 21 days before the grass. Sowing both species at the same time resulted in a dominant grass sward, with R. obtusifolius becoming dominant when sown 42 days prior to L. perenne. Grass sowing rate had no effect on R. obtusifolius growth or biomass. A second experiment investigated how competition from L. perenne sown 21 days after R. obtusifolius combined with damage from G. viridula and/or U. rumicis (applied at either the 3-4- or 10-13-leaf stage, or at both stages) affected the growth and final biomass of R. obtusifolius. Beetle grazing at the latter leaf stage was the only treatment that reduced R. obtusifolius biomass, although rust infection at the earlier application led to an increase in shoot and root weight. The results are discussed in terms of the potential for use of these agents in the field.

The biocontrol fungus pochonia chlamydosporia shows nematode host preference at the infraspecific level

A RAPD-PCR assay was developed and used to test For competitive variability in growth of the nematode biological control fungus Pochonia chlamydosporia. Saprophytic competence in soil with or without tomato plants was examined in three isolates of the fungus: RES 280 (J), originally isolated from potato cyst nematode (PCN) cysts; RES 200 (1) and RES 279 (S), both originally isolated from root knot nematode (RKN) eggs. Viable counts taken at 70 d indicated that I was the best saprophyte followed by S, with J the poorest. RAPD-PCR analysis of colonies from mixed treatments revealed that there was a cumulative effect of adding isolates to the system. This Suggested that the isolates did not interact and that they may occupy separate niches in soil and the rhizosphere. To investigate parasitic ability, soils were seeded with two isolates of the fungus: J and S, singly or in combination. Tomato or potato plants were grown in these soils; free of nematodes, or inoculated with PCN or RKN, and incubated for 77 d. The abundance of the PCN isolate J in PCN cysts was significantly greater than that of the RKN isolate S but in RKN egg masses, S was significantly more abundant than J. RAPD-PCR analysis of colonies from mixed treatments confirmed that J was more abundant than S ill PCN cysts whereas the converse was observed on RKN egg masses. This substantiates the phenomenon of nematode host preference at the infraspecific level of P. chlamydosporia and highlights its relevance for biological control of plant parasitic nematodes.

Effect of water stress on infection by species of honey fungus (Armillaria mellea and A. gallica)

Isolates of Armillaria mellea and A. gallica that differed in virulence to healthy blackcurrant, strawberry, Lawson cypress and privet were used to inoculate plants exposed to different watering regimes. Host plants from which water had either been withheld or their roots kept constantly flooded with water, both showed increased susceptibility compared to those plants, which had been watered regularly. At the end of the period of stress, roots from randomly selected plants from each treatment were harvested. Following chemical analysis of the roots for protein, lipids, and carbohydrates including starch, in vitro assays were carried out with these substances. The increased amounts of these nutrients in both groups of stressed plants are sufficient to stimulate the growth of both A. mellea and A. gallica and enhance their virulence.

Oxidation affects the flow-induced aggregation of low density lipoprotein and its inhibition by albumin

We investigated whether oxidation alters the self-aggregation of low density lipoprotein (LDL) and the inhibition of such aggregation by albumin. Incubation with copper for different durations produced mildly, moderately, and highly oxidised LDL (having, respectively, ca. 60, 300 and 160 nmol lipid hydroperoxides/mg protein, and electrophoretic mobilities 1.2, 2.6 and 4.4 times that of native LDL). The rate of flow-induced aggregation was the same for native, mildly oxidised and moderately oxidised LDL, but decreased for highly oxidised LDL. The inhibitory effect of albumin (40 mg/ml) on aggregation was reduced by mild oxidation and further reduced by moderate or severe oxidation. The net result of the two effects was that in the presence of albumin, moderately oxidised LDL had the highest rate of aggregation and native the lowest. The reduction in the anti-aggregatory effect of albumin provides a new mechanism by which LDL oxidation might enhance net aggregation in vivo. (C) 2003 Elsevier B.V. All rights reserved.